In this study, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 mo) and old (30 mo) FXBN rats. Using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE), we have obtained 2-D gel maps of 301 identified non-redundant proteins from rat aorta and observed 18 proteins that significantly change abundance with aging. Utilizing Isobaric tags for relative and absolute quantitation (iTRAQ), 921 proteins were quantified and between both methods, 50 proteins were shown to have significantly different age-associated abundance. Proteomic analysis shows that one protein of interest, milk fat globule protein E8 (MFG-E8), significantly increases in abundance in old rat aortae. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species, including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within vascular smooth muscle cells (VSMC) of the thickened aged aortic wall. Exposure of early passage VMSC from young aorta to Ang II markedly increases MFG-E8 and enhances the invasive capacity to levels observed in VSMC from old rats. Treatment of VSMC with MFG-E8 increases MCP-1 and VSMC invasion that are inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMC invasion capacity. Thus, arterial MFG-E8 significantly increases with aging and is a key component within the Ang II MCP-1/VSMC invasion signaling cascade. We have identified that aging arterial MFG-E8-enriched VSMC are activated and proliferating both in vivo and in vitro. Increased MFG-E8 in VSMC triggers phosphorylation of extracellular signalregulated kinases-1/2(ERK-1/2), augments levels of proliferation cell nuclear antigen (PCNA) and cyclin-dependent kinase 4 (CDK4), increases BrdU incorporation and promotes cell growth. The knockdown of MFG-E8 reduces the rate of cell cycling, accelerating signaling molecules PCNA and CDK4 expression, facilitating cell entry into a growth-arrested state. Furthermore, we find that integrin v5 and platelet derived growth factor (PDGF) receptor are upregulated with MFG-E8 and also are elements to relay proliferative signals to aging VSMC. In addition, MFG-E8 facilitates the profibrosis of VSMC with aging via an interaction with caveolin 1 (CAV1) signaling. Exposure of isolated aortic VSMC of old rats to MFG-E8 increases the profibrogenic signaling molecules TGF-1, TGF- receptor type II (TRII), p-SMAD (Sma and Mad: Mothers against decapentaplegic)2/3, and collagen I (Col I) while knockdown of MFG-E8 gene expression substantially reduces the expression of these molecules. Both co-immuno-labeling and co-immune-precipitation of MFG-E8, TRII, and CAV1 in young VSMC (8-mo) indicates their protein-protein interactions. In young VSMC, MFG-E8 up-regulates the expression of CAV1. Knockdown of the CAV1 gene, similar to MFG-E8 exposure, significantly increases the expression of TGF-1, TRII, p-SMAD-2/3, and Col I. Over-expression of the CAV1 gene, similar to MFG-E8 gene knockdown, markedly decreases the expression of these fibrogenic molecules in both young and old VSMC. Furthermore, MFG-E8 treatment significantly increases the expression of TGF-1, p-SMAD-2/3, and Col I in CAV1 silenced young VSMC. Interestingly, MFG-E8 exposure does not increase expression of these profibrogenic molecules in old VSMCs when CAV1 is overexpressed. These results, for the first time, demonstrate that MFG-E8 modulates the TGF-1 fibrogenic signaling in VSMC in a CAV1/age-dependent manner. Importantly, we find that the involvement of MFG-E8 in atherosclerotic lesions increases with age. In in vivo studies: 1) Immunostaining demonstrates that MFG-E8 protein in the aortic wall in ApoE-/- mice on a standard chow diet increases with age and is localized predominantly in advanced plaques and the elastin laminae . 2) In nonhuman primates (9 to 23- years old), iTRAQ assay indicates that levels of arterial MFG-E8 protein are highly correlated with age. In older monkeys (>17-years old), that were fed a high cholesterol diet for 2 years, a 1.6 fold increase was seen in the amount of arterial MFG-E8 protein and its fragment medin that was expressed when compared to older animals fed a standard diet.3) In aortic thoracic intimae isolated from adult humans (age 22-to 69-years old), Western blot analysis indicates that the abundance of aortic MFG-E8 protein and its fragment medin significantly increased with advancing age; 4) Immunostaining indicates that MFG-E8 is mainly derived from smooth muscle cells (alpha-smooth actin positive) and macrophages (CD 68 positive). In in vitro studies: 1) Early passage thoracic aortic smooth muscle cells (SMC) from both young and old monkeys treated with pathogenic oxidative low density lipoprotein showed that the increase in the uptake capacity of oxLDL in old cells exceeded that of young cells, thus facilitating the transdifferentiation of the SMC into foam-like cells (CD68 positive). 2) Silencing of MFG-E8 RNA markedly reduced the uptake of oxLDL by young and old monkey SMC. In addition, an MFG-E8 fragment, medin, plays an important role in arterial aging and atherosclerosis. The effects of medin on VSMCs proinflammation with advancing age are still under investigation. Taken together, MFG-E8 translation and glycosylation and its fragment medin are increased in atherosclerotic aortic walls with aging in various species, including humans, potentially playing a precipitating role in the formation of foam cells, the hallmark of atherosclerosis.